The objectives of the proposed research are to identify the genes and gene products which regulate hemoglobin biosynthesis and to develop methods for selectively stimulating human fetal hemoglogin production. Stimulation of fetal hemoglobin production is a therapeutic approach to many of the beta hemoglobinopathies, e.g. sickle cell anemia, and to beta thalassemia. Hemoglobin synthesis will be studied in cultured animal cells. Continuous cell lines which synthesize hemoglobin have been isolated from mice and rats with erythroleukemia. Some of these cell lines show a requirement for drug (DMSO and others) induction of hemoglobin synthesis. Methods are available to quantitate the production of specific hemoglobins, specific globin chains, heme, globin chain mRNA, heme biosynthetic enzymes, and red cell surface antigens. These methods include incorporation of radioactive precursors, electrophoresis, chromatography, DNA-RNA hybridization, standard enzyme assays, and immunologic techniques. Various biochemical parameters such as cyclic AMP, cyclic GMP and RNA polymerases will be measured in induced and uninduced cultured cells. Stimulatory or inhibitory erythropoietic factors will be sought. Genetically variant cultured mouse cells for biochemical analysis will be sought from intact animals and by in vitro mutagenesis. For optimum studies of stimulation of human fetal hemoglobin production, attempts will be made to isolate human cell lines which produce hemoglobin in continuous culture. Primary isolates from bone marrow of patients with leukemia and polycythemia vera will be used as well as primary isolates from fetal erythropoietic tissue. Mutagenesis and alternate passage in vitro and in vivo in animals will be used to isolate human hemoglobin producing cell lines. Human primary bone marrow cultures will also be used for regulation studies of fetal hemoglobin production. The effect of various biochemical compounds and tissue extracts on the relative amount of fetal, embryonic and adult hemoglobin will be examined in cultured mouse and human cells.